Functional Study On Tweedle Genes From Locusta Migratoria And The Regulation Mechanism Of Chitin Metabolism

The insect cuticle provides the primary barrier for insect defence againstchangeable environment condition.Along with many advantages,the cuticlealso makes the arthropod's life more complicated:to accommodateenvironment changes during the life cycle and to allow growth to the nextdevelopmental stage,the cuticle has to be shed and replaced by a new oneduring development.The cuticle is produced from cuticular proteins andchitin via a series of biochemical reactions.Tweedle proteins are important members of the cuticular proteinfamily,which have four conserved motifs binding to chitin.TweedleDmutation leads to insufficient cuticle thickness and failure to maintain anormally slender shape,ultimately resulting in a squat body shape,suggesting Tweedle family genes play a profound effect on cuticledevelopment.Here,we reported that the cuticular protein genes---LmTwdl1and LmmTwdl2 of Locusta migratoria belong to the Tweedle family.Todetermine the tissue-specific and developmental expression patterns ofTweedle family genes,we performed real-time PCR assays in the fifth-instarstage.The results found that the transcription level of Tweedle genes washigh in the integument and foregut,but low in the midgut,hindgut,brain,gastric caeca and Malpighian tubules.The developmental expression patternsrevealed low expression in the cuticle during the early and middle stages ofthe fifth-instar nymphs;in contrast,its expression rapidly increased in the latestages of fifth-instar nymphs.In situ hybridization showed that LmTwdl1 islocalized to epidermal cells of the cuticle.We then performed RNAinterference(RNAi)to examine the function of Tweedle genesin locusts.Silencing of LmTwdll resulted in high mortality during moulting processbefore the next stage.Although there were no obvious difference betweenRNAi LmTwdl1 and the control group both in integument construct and chitin distribution of the old cuticle or the newly synthesized cuticle.Electronmicroscopic examination showed the epicuticle of nymphs failed to molttended to be thinner,andthe arrangement of chitin in the procuticleappears tobe disordered compare to the control group.These results demonstrate thatLmTwdll plays a critical role in moulting,which contribute to a betterunderstanding of the distinct functions of the Tweedle family in locusts.The roles of chitin synthetase and chitinases,as two crucial enzymes,have been recognized in the chitin biosynthesis and degradation during insectmolting.miR-71 and miR-263 can suppress LmCHSl and LmCHTIOtranscript and control molt development by precisely regulate chitinmetabolism.We 1)detected the expression levels of LmCHS1 and LmCHT10after miRNA agomir(overexpression)administration in the locust integumentto determine the effects of miR-71 and miR-263 on their target genes in vivo;2)performed in situ analyses of miRNA-71/miR-263 and their targets bymiRNA/mRNA fluorescence in situ hybridization to unveil whethermiR-71/miR-263 was co-localized in the locust integument.3)performed theHematoxylin and eosin(H&E)stain and chitin stain in the integument byinjecting agomir-71 or agomir-263 into the locusts to further determine if theabnormally layer of the cuticle was responsible for the molting defectinduced by miR-71 and miR-263.These miRNAs-mediatedpost-transcriptional regulation mechanism of chitin metabolism isparticularly significant for understanding the molt development of locustsand potentially provides new targets for controlling locust plagues worldwide.